Iris as a recipient tissue for pigment cells: Organized in vivo differentiation of melanocytes and pigmented epithelium derived from embryonic stem cells in vitro

Regenerative transplantation of embryonic stem (ES) cell-derived melanocytes into adult tissues, especially skin that includes hair follicles or the hair follicle itself, generally not possible, whereas that of ES cell-derived pigmented epithelium was reported previously. We investigated the in vivo differentiation of these two pigment cell types derived from ES cells after their transfer into the iris. Melanocytes derived from ES cells efficiently integrated into the iris and expanded to fill the stromal layer of the iris, like those prepared from neonatal skin. Transplanted pigmented epithelium from either ES cells or the neonatal eye was also found to be integrated into the iris. Both types of these regenerated pigment cells showed the correct morphology. Regenerated pigment epithelium expressed its functional marker. Functional blocking of signals required for melanocyte development abolished the differentiation of transplanted melanocytes. These results indicate successful in vivo regenerative transfer of pigment cells induced from ES cells in vitro. Developmental Dynamics 237:2394-2404, 2008. (c) 2008 Wiley-Liss, Inc.     

Fiber arrangements of nerves belonging to ventral and dorsal divisions in the proximal region of the brachial plexus: a study using fluorescence of DiI and DiO in adult rats

The motor and sensory fiber arrangements in the proximal region of the spinal nerves are important for understanding the relationship of the peripheral nerves to neuron distribution. On the other hand, the fiber arrangements are also important for the treatment of peripheral nerve grafting. We studied the fiber arrangements of two divisions (ventral and dorsal) in the proximal region of the brachial plexus and the fiber arrangements of the lateral cutaneous rami in Th7 and Th8 intercostal nerves in adult rats with a method using the fluorescent pigments DiI and DiO. Results showed that fiber arrangements belonging to the two divisions have a specific separate distribution in the distal region. However, this specific separate distribution form was absent in the proximal region of the spinal nerves in the plexus. Fiber arrangements of the lateral cutaneous ramus in the anterior branches of the thoracic nerves (intercostal nerves) were also observed in comparison with those in the brachial plexus by the same method. In the intercostal nerves, fibers of the lateral cutaneous ramus were distributed in the dorsal portion from distal to proximal. These results suggest that there are two types of fiber arrangement in the proximal regions of the spinal nerves: a ventrodorsal distributional type, comprising intercostal nerves and the dorsal branches of the spinal nerves; and a mixed type, comprising the anterior branches of the cervical and brachial (and perhaps lumbar) plexuses. On the other hand, fibers of the lateral cutaneous rami in the intercostal nerves were distributed on the dorsal part of the nerves. These results of fiber arrangement analysis for the intercostal nerves may offer an opportunity to improve the effect of treatments using peripheral nerve grafting and suturing in the brachial and lumbar plexus with intercostal nerves.     

Absence of SHATI/Nat8l reduces social interaction in mice

Rapidly adapting mechanically activated channels (RA) are expressed on somatosensory neurons and thought to play a role in mechanical transduction. Because mechanical sensations can be significantly affected by temperatures, we examined thermal sensitivity of RA currents in cultured dorsal root ganglion (DRG) neurons to see if RA channel activity is highly temperature-dependent. RA currents were evoked from DRG neurons by membrane displacements and recorded by the whole-cell patch-clamp recording technique. We found that RA currents were significantly enhanced by warming temperatures from 22 to 32 °C and reduced by cooling temperatures from 24 to 14 °C. RA channel activation exhibited steep temperature-dependence with a large temperature coefficient (Q10>5) and a high activation energy (Ea>30 kcal/mol). We further showed that RA channel activation by mechanical stimulation led to membrane depolarization, which could result in action potential firing at 22 °C or 32 °C but not at 14 °C. Taken together, our results provide the measurements of thermal dynamics and activation energy of RA channels, and suggest that a high energy barrier is present for RA channels to open. These findings are in agreement with temperature sensitivity of mechanical sensations in mammals.     

Two distinct lymphocyte homing systems involved in the pathogenesis of chronic inflammatory gastrointestinal diseases

Under normal and pathological conditions, lymphocyte migration into the gastrointestinal mucosa to form gut-associated lymphoid tissue is mediated by the L-selectin ligand peripheral lymph node addressin and the integrin α4β7 ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expressed on high endothelial venules (HEVs) and HEV-like vessels. In this review, we discuss these two distinct lymphocyte homing systems involved in the pathogenesis of chronic inflammatory gastrointestinal diseases with reference to our and others’ previously published works. We also describe a recently developed recombinant integrin α4β7 heterodimeric IgG chimera that can be used as an immunohistochemical reagent to stain functional MAdCAM-1.     

Perinatal outcomes for transfer of blastocysts vitrified and warmed in defined solutions with recombinant human albumin: 374 babies born after 898 embryo transfers

Purpose

To assess the efficacy of a novel, defined vitrification procedure using recombinant human albumin (rHA) for cryopreservation of human blastocysts. Design: Retrospective study. Setting: Private IVF clinic. Patients: 1,496 patients received vitrified/warmed embryo transfer (ET).

Methods

Surplus blastocysts, and blastocysts from patients undergoing elective embryo cryopreservation, were vitrified/warmed using Cryotop carriers in homemade solutions containing either human serum albumin (HSA) or rHA. Main Outcome Measures: Clinical and neonatal outcomes regarding the vitrified/warmed ET procedures.

Results

The HSA and rHA groups had a total of 1,163 and 898 vitrified/warmed cycles, respectively. Embryo survival rates (98.7 % vs. 98.9 %, respectively) and the number of embryos transferred (1.08 ± 0.01 vs. 1.06 ± 0.01, respectively) were similar in the HSA and rHA groups. Clinical pregnancy rates/ET were higher (P < 0.05) in the rHA group (56.0 %) than in the HSA group (51.5 %). The HSA and rHA groups had similar live delivery rates/pregnancy (72.2 % vs. 72.3 %, respectively) and perinatal outcomes, including birth weight (2,988 ± 28 vs. 3,046 ± 26 g, respectively). Birth defects occurred in 0.9 % and 1.6 % of neonates in the HSA and rHA groups, respectively.

Conclusions

rHA effectively replaced HSA for human embryo vitrification procedures, and yielded high rates of pregnancy and live births after vitrified/warmed ET. This new approach will support the development of defined ART systems, which will eliminate the variation and risks associated with the use of blood-derived products.     

Seroprevalence of Kaposi's sarcoma‐associated herpesvirus among men who have sex with men in Japan

Kaposi's sarcoma‐associated herpesvirus (KSHV), the etiologic agent of Kaposi's sarcoma, causes malignancies frequently in patients with acquired immunodeficiency syndrome. In the United States and Europe, KSHV infection is common among men who have sex with men. However, the seroprevalence of KSHV among men who have sex with men in Japan is unknown. In the present study, the seroprevalence of KSHV was investigated among 230 men who have sex with men and 400 age‐ and area of residence‐matched men (controls) using a mixed‐antigen (KSHV‐encoded K8.1, open reading frame 59, 65, and 73 proteins) enzyme‐linked immunosorbent assay and an immunofluorescence assay. Among the Japanese men who have sex with men, serological assays revealed that 27 (11.7%) were seropositive for KSHV; 20 (5%) of the men in the control group were also KSHV seropositive. The seroprevalence of KSHV among men who have sex with men was significantly higher than in the control group (odds ratio = 2.52, 95% confidence intervals = 1.38–4.62, P = 0.0019, Chi‐square test). Infection with the human immunodeficiency virus, Treponema pallidum, or hepatitis B and C virus did not correlate with KSHV infection. Furthermore, the association of KSHV seropositivity with specific sexual activities was not statistically significant. In conclusion, a higher KSHV seroprevalence was found among Japanese men who have sex with men than among the controls, suggesting that the circulation of KSHV infection is more efficient among men who have sex with men in Japan than among men who do not engage in such sexual activities. J. Med. Virol. 85: 1046–1052, 2013. © 2013 Wiley Periodicals, Inc.     

Keratinocyte Differentiation and Proliferation are Regulated by Adhesion to the Three-Dimensional Meshwork Structure of Type IV Collagen

We examined the behavior of human foreskin keratinocytes (HFKs) on reconstituted type IV collagen gel. HFKs survived for several days and the upper layer cells expressed a differentiation marker, involucrin. Apoptosis was induced after involucrin expression while cell proliferation was suppressed. On molecular type IV collagen, integrins shifted from α2β1 to α3β1 during HFK culture. On type IV collagen gel, HFKs initially expressed integrin α2β1, and later expressed integrin α3β1 in the presence of α2β1 did not disappear. Using synthetic peptides, we examined integrin α2-mediated adhesion to type IV collagen gel. Addition of synthetic peptide dose-dependently inhibited cell adhesion both on type IV collagen gel and on molecular type IV collagen. On type IV collagen gel, weaker phosphorylation of focal adhesion kinase, paxillin, and Akt was observed compared with the molecular forms. Based on these observations, we think type IV collagen gel is a novel culture substrate that mimics the physiological environment for HFKs.     

Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line

Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs. Tailored libraries were first constructed by replacing endogenous immunoglobulin genes with designed human counterparts. From these libraries, clones producing full-length human IgGs against distinct antigens can be isolated, as exemplified by the selection of antagonistic mAbs. Taking advantage of avian biology, effective affinity maturation was achieved in a straightforward manner by seamless diversification of the parental clones into secondary libraries followed by single-cell sorting, quickly affording mAbs with improved affinities and functionalities. Collectively, we demonstrate that the human ADLib system could serve as an integrative platform with unique diversity for rapid de novo generation and optimization of therapeutic or diagnostic antibody leads. Furthermore, our results suggest that libraries can be constructed by introducing exogenous genes into DT40 cells, indicating that the ADLib system has the potential to be applied for the rapid and effective directed evolution and optimization of proteins in various fields beyond biomedicine.